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Mathijsen started his football career at SV VOAB in Goirle after he visited a training session of a friend as a young boy. At the age of nine he left this club and came through the youth system of Willem II Tilburg. He was promoted to the first team for the 1998–99 season, making his debut on 27 February 1999 in a match against FC Utrecht. After six seasons at Willem II, he was sold by manager Co Adriaanse to AZ Alkmaar. However he endured a harder time breaking into the first team at Alkmaar and his case was not helped by injuries.

After finishing as runners-up in the Eredivisie in 2006, Mathijsen opted to transfer to German Bundesliga side Hamburg for €6 million where he teamed up with fellow Dutchmen Rafael van der Vaart and Nigel de Jong. He scored Hamburg's 2500th Bundesliga goal (his first for the club) on 20 October 2007 against Stuttgart.[3]

In November 2008, Mathijsen signed a new contract extension, tying him to Hamburg until the summer of 2012.[4]

In mid June 2011, however, he signed a two-year contract with La Liga side Málaga CF.

In the summer of 2012, it became apparent that Malaga was willing to let Mathijsen transfer to another club, and several Dutch sides have shown interest, including Feyenoord and Ajax. On 10 August, Mathijsen signed a contract at Dutch side Feyenoord.

On 17 November 2004, Mathijsen made his international debut for the Netherlands against Andorra in a 3–0 away win.

Since his international debut, Mathijsen has become a mainstay figure in the national team, especially after impressing at Hamburg. He is usually positioned as left central defender, because of his strong left-foot. He was called up to the Dutch squad for Euro 2008 in Austria and Switzerland. Initially in danger of losing his spot to Wilfred Bouma, he followed it up with a decent enough performance at the Euro 2008 and remained first choice.[5]

Mathijsen was ever present for Oranje during the 2010 World Cup qualifying.[6] He played more minutes than any other player in the Dutch squad and played a key role in their record of having conceded the fewest goals in their qualifying group.